The paper presents the study of anisotropic fluorescence decay of FAD in water-methanol solutions of different concentrations in the range of 0-80%. The fluorescence decay parameters: decay times, corresponding weighting coefficients, fluorescence anisotropy, and rotation diffusion time have been determined from experiment using the TCSPC method as function of methanol concentration and analyzed. The fluorescence kinetics demonstrated doubleexponential decay with two fluorescence decay times of about 2 and 4 ns. The decay times τ1 and τ2 were found to be the same under excitation at 450 nm and at 355 nm within experimental error bars and were practically independent of methanol concentration. The fluorescence anisotropy under 450 nm and 355 nm excitation was determined to be about 0.35 and 0.23, respectively indicating that the directions of the excitation transition dipole moments via the first and the second absorption bands differed significantly from each other. Also, the anisotropy was found to be practically independent of methanol concentration. The rotational diffusion time was proportional to the solution viscosity at lower methanol concentrations up to 40%, and reached a plateau at higher concentrations while the solution viscosity dropped down. This behavior was explained due to the increase of FAD solvation in the MeOH-water solution under the condition of FAD unfolding due to the denaturation effect.
The influence of solution polarity and viscosity on fluorescence decay parameters in free and protein-bound NADH was studied. The fluorescence in NADH dissolved in water, methanol, ethanol, propylene glycol, and alcohol dehydrogenase-containing solution was two-photon excited by femtosecond laser pulses at 720 nm and recorded by means of the time-correlated single photon counting (TCSPC) method. Fluorescence decay times and corresponding weighting coefficients were determined by fit from polarization-insensitive fluorescence decay experimental signals. The fluorescence decay times τ1 and τ2, and weighting coefficients a1 and a2 were found to depend significantly on the solution type. A model describing the dependence of the fluorescence decay parameters on the microenvironment in solutions with different polarity and viscosity has been developed. According to the model, the heterogeneity in the measured fluorescence decay times in NADH and the time values were closely related with the charge distributions in the cis and trans configurations of the nicotinamide ring that result in different electrostatic field and different non-radiative decay rates. The influence of solution polarity and viscosity on the measured fluorescence decay times was investigated. As shown in high viscous solutions the increase of fluorescence decay times in NADH was mostly due to slowing down of the nuclei motions during the vibrational relaxation and intramolecular nuclear rearrangement whereas in low viscous solutions the fluorescence decay times follow the change of solution polarity.
A novel polarization-modulation transient method has been developed for studying fast anisotropic relaxation in electronic excited states of polyatomic and biologically relevant molecules under excitation with femtosecond laser pulses. The method is based on the modulation of pump beam polarization with a photo-elastic modulator and detection of an anisotropic contribution to the transient signal by a highly-sensitive demodulation balanced scheme. The method was tested on aqueous solution of coenzyme NADH (nicotinamide-adenine-dinucleotide) pumped at 360 nm and probed at 720 nm. Anisotropic vibrational relaxation and rotational diffusion have been observed in the sub-picosecond time domain. The method significantly enhances the accuracy of transient measurements and allows for recording of high-quality signals at low energy (a nJ) pump pulses.
We studied polarized fluorescence in biological coenzyme NADH in water-methanol solutions upon two-photon excitation with femtosecond laser pulses at 720 nm. The polarized fluorescence decay was recorded by a time correlated single photon counting (TCSPC) system. Fluorescence decay times, rotational diffusion time, fluorescence anisotropy, and the ratio of two pre-exponential factors have been determined and studied as a function of methanol concentration. The results obtained were interpreted on the basis of a model of NADH denaturation processes in solutions and can be used for modeling of NADH binding with various dehydrogenases in living cells.
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